Control of Hedgehog Signal Transduction by Neuropilin

描述（由申请人提供）：在大多数发育中的组织和器官中，Hedgehog （Hh）信号被用于控制细胞命运，以及许多再生事件。Hh信号的缺陷会导致出生缺陷和癌症。关于Hh信号是如何转导的，仍然存在许多机制上的谜团。通过高通量RNAi筛选，我们鉴定出Neuropilins (Nrp) 1和2是脊椎动物Hh信号传导的新型特异性调节因子。Nrps是一种单遍跨膜蛋白，参与多种分泌配体的接受，包括信号蛋白和VEGF165，并参与细胞粘附和细胞迁移。在成纤维细胞中，阻断Nrps对Hh信号转导的抑制作用与阻断纤毛形成或阻断平滑功能的作用一样强。相反，任何一种Nrp的过量产生都会使细胞对Hh信号敏感。Hh通路的新成分很少被发现；Nrps可能由于部分冗余而被遗漏。我们发现的这两种蛋白的功能是这一重要的形态发生途径所必需的，它们有可能从根本上改变目前的Hh信号传导模型，并扩大对其他信号传导途径中Nrp功能的理解。目的1。确定Nrps调控Hh信号转导的机制。Nrps可以通过直接与已知的Hh通路组分相关联，或介导与Hh转导汇合的其他信号，或通过改变Hh转导所需的细胞特性或过程来影响Hh信号转导。我们将通过确定Hh信号传导的哪些步骤受到影响，Nrps对Hh信号传导的影响是否涉及细胞粘附或迁移变化，以及哪些蛋白质直接与Nrps相互作用来研究每种可能性。目标2。确定哪些Nrp结构域需要支持Hh信号传导，以及已知的Nrp共受体、配体或效应分子是否能够交叉调节Hh通路。我们将以两种方式研究哪些结构域有助于Hh信号转导：工程结构域缺失，以及高通量筛选干扰Nrp支持Hh信号转导的点突变。Nrps转导VEGF和Semaphorin信号，分别作为VEGF受体和丛蛋白的共受体。我们将测试VEGF、VEGF受体、丛蛋白受体或信号蛋白是否参与Nrps对Hh信号转导的影响。目标3。研究Nrps如何影响Hh依赖性的发展和肿瘤发生。使用携带两个Nrp基因突变的小鼠，我们将控制Nrp功能的时间和组织特异性去除，以测试它们在体内参与几种依赖hh的发育过程。使用新创建的慢病毒，编码特异性抑制rna，阻断nrp基因，我们将感染Hh应答细胞的原代培养物，并监测对Hh靶基因表达的影响。

DESCRIPTION (provided by applicant): Hedgehog (Hh) signaling is employed in controlling cell fates in most developing tissues and organs, as well as during many regeneration events. Defects in Hh signaling lead to birth defects and cancer. Many mechanistic mysteries remain regarding how an Hh signal is transduced. Using high-throughput RNAi screening, we identified Neuropilins (Nrp) 1 and 2 as novel, specific regulators of vertebrate Hh signaling. Nrps are single-pass transmembrane proteins implicated in the reception of a diverse set of secreted ligands, including Semaphorins and VEGF165 and in cell adhesion and cell migration. In fibroblasts the inhibition of Hh signal transduction resulting from blocking Nrps is as strong as the effect of blocking cilia formation or blocking Smoothened function. Conversely, over-production of either Nrp sensitizes cells to Hh signals. New components of the Hh pathway are uncovered infrequently; the Nrps were probably missed due to their partial redundancy. Our discovery of two proteins whose functions are required by this important morphogenic pathway has the potential to bring fundamental changes to current models of Hh signaling and to enlarge the understanding of Nrp functions in other signaling pathways. Aim 1. Determine the mechanism by which Nrps regulate Hh signal transduction. Nrps could influence Hh signal transduction by directly associating with known Hh pathway components, or mediating other signals that converge with Hh transduction, or by altering cell properties or processes that are required for Hh transduction. We will investigate each of these possibilities by determining which steps in Hh signaling are affected, whether cell adhesion or migration changes are involved in the effect of Nrps upon Hh signaling, and what proteins interact directly with Nrps. Aim 2. Determine which domains of Nrp are needed to support Hh signaling, and whether known Nrp co-receptors, ligands, or effector molecules are capable of Hh pathway cross- regulation. We will investigate which domains contribute to Hh signal transduction in two ways: engineered domain deletions, and a high-throughput screen for point mutations that interfere with Nrp support of Hh signal transduction. Nrps transduce VEGF and Semaphorin signals, acting as co-receptors for VEGF receptors and Plexins, respectively. We will test whether VEGF, VEGF receptor, Plexin receptors, or Semaphorins are involved in the effect of Nrps upon Hh signal transduction. Aim 3. Investigate how Nrps influence Hh- dependent development and tumorigenesis. Using mice that carry mutations in both Nrp genes, we will control temporal and tissue-specific removal of Nrp functions to test their involvement in several Hh-dependent developmental processes in vivo. Using newly created lentiviruses, which encode specific inhibiting RNAs that block the nrp genes, we will infect primary cultures of Hh-responsive cells and monitor effects on Hh target gene expression.